Increased Serum Levels of Anti-Carbamylated 78

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Sep 8, 2016 - with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and primary ... peptides (anti-CCP), anti-GRP78, and anti-CarGRP78 were ...
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Molecular Sciences Article

Increased Serum Levels of Anti-Carbamylated 78-kDa Glucose-Regulated Protein Antibody in Patients with Rheumatoid Arthritis Hui-Chun Yu 1 , Pei-Hsuan Lai 2 , Ning-Sheng Lai 2,3 , Hsien-Bin Huang 4 , Malcolm Koo 1,5 and Ming-Chi Lu 2,3, * 1 2 3 4 5

*

Department of Medical Research, Dalin Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Dalin, Chiayi 62247, Taiwan; [email protected] (H.-C.Y.); [email protected] (M.K.) Division of Allergy, Immunology and Rheumatology, Dalin Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Dalin, Chiayi 62247, Taiwan; [email protected] (P.-H.L.); [email protected] (N.-S.L.) School of Medicine, Tzu Chi University, Hualien 97004, Taiwan Department of Life Science and Institute of Molecular Biology, National Chung Cheng University, Minxiong, Chiayi 62102, Taiwan; [email protected] Dalla Lana School of Public Health, University of Toronto, Toronto, ON M5T 3M7, Canada Correspondence: [email protected]; Tel.: +886-5264-8000 (ext. 3205)

Academic Editor: Anastasia Susie Mihailidou Received: 10 August 2016; Accepted: 5 September 2016; Published: 8 September 2016

Abstract: The objective of this study was to investigate the presence and titer of anti-carbamylated 78-kDa glucose-regulated protein (anti-CarGRP78) antibody in serum from controls, and patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and primary Sjögren syndrome (pSS). Thirty-three RA patients, 20 SLE patients, 20 pSS patients, and 20 controls were enrolled from our outpatient clinic. GRP78 was cloned and carbamylated. Serum titers of anti- cyclic citrullinated peptides (anti-CCP), anti-GRP78, and anti-CarGRP78 were measured with an enzyme-linked immunosorbent assay. No differences in serum titers of anti-GRP78 antibody in patients with RA, SLE, or pSS compared with the controls were observed. Serum levels of anti-carGRP78 antibody in patients with RA, but not SLE or pSS, were significantly higher compared with the controls (OD405 0.15 ± 0.08 versus 0.11 ± 0.03, p = 0.033). There was a positive correlation between the serum levels of anti-GRP78 antibody, but not anti-CarGRP78 antibody, with the levels of anti-CCP antibody in patients with RA. Both anti-GRP78 and anti-carGRP78 antibodies failed to correlate with C-reactive protein levels in patients with RA. In conclusion, we demonstrated the presence of anti-CarGRP78 antibody in patients with RA. In addition, the serum titer of anti-CarGRP78 antibody was significantly elevated in patients with RA compared with the controls. Anti-CarGRP78 antibody could also be detected in patients with SLE or pSS. Keywords: GRP78; autoantibodies; rheumatoid arthritis; systemic autoimmune diseases

1. Introduction The 78 kDa glucose-regulated protein (GRP78), also known as binding immunoglobulin protein (BiP), has been shown to be an important autoantigen for rheumatoid arthritis (RA). GRP78 could stimulate the proliferation of synovial T cells from patients with RA [1]. Anti-GRP78 antibody can be found in the serum from patients both after and before their diagnosis of RA [2]. Anti-citrullinated protein antibody (ACPA) is known to be a very specific diagnostic marker for RA. In our previous study, we found that GRP78 was citrullinated (citGRP78) and became recognizable by ACPAs, and ACPAs could directly stimulate monocytes to secrete tumor necrosis factor α (TNF-α) via binding to citGRP78 [3]. In addition, anti-citGRP78 antibody was also widely detected in serum from patients

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withRA RA[4]. [4].Moreover, Moreover,parenteral parenteraladministration administrationofofGRP78 GRP78could couldalso alsoameliorate amelioratejoint jointinflammation inflammation with inmice micewith withcollagen-induced collagen-inducedarthritis arthritis[5]. [5]. in Carbamylation is a post-translational or Carbamylation is a post-translational modification modification in inwhich whichcyanate cyanatebinds bindstotoprimary primaryamino amino thiol groups. Recently, autoantibodies recognizing carbamylated proteins (anti-CarP antibodies) or thiol groups. Recently, autoantibodies recognizing carbamylated proteins (anti-CarP antibodies) werefound foundto tobe bepresent presentin inthe thesera serafrom frompatients patientswith withRA RAand andwere werepredictive predictivefor for joint joint damage damage [6]. [6]. were In addition, the presence of anti-CarP antibodies could predict the development of RA [7] and serve In addition, the presence of anti-CarP antibodies could predict the development of RA [7] and serve as a poor prognostic for long-term disability in patients with [8]. Although initial aas poor prognostic factorfactor for long-term disability in patients with RA [8]. RA Although the initialthe antigen antigen used for detecting anti-CarP antibodies was carbamylated fetal calf serum [6], other used for detecting anti-CarP antibodies was carbamylated fetal calf serum [6], other carbamylated carbamylated antigens such as α-enolase or vimentin [9,10] were found to be part of the anti-CarP antigens such as α-enolase or vimentin [9,10] were found to be part of the anti-CarP antibodies. It is antibodies. that It isother conceivable other proteins could carbamylated and recognizable by conceivable proteinsthat could be carbamylated andberecognizable by anti-CarP antibodies. anti-CarP antibodies. Therefore, we hypothesized that the anti-CarGRP78 antibody might be present Therefore, we hypothesized that the anti-CarGRP78 antibody might be present in the serum from in the serum fromThe patients The serum titers of anti-CarGRP78 antibody patients patients with RA. serumwith titersRA. of anti-CarGRP78 antibody among patients with among other systemic with other systemic autoimmune diseases, systemic erythematosus (SLE) and autoimmune diseases, including systemic lupusincluding erythematosus (SLE)lupus and primary Sjögren syndrome primary Sjögren syndrome (pSS), were also investigated in this study. (pSS), were also investigated in this study. 2.2. Results 2.1. 2.1.Patients Patientsand andControls Controls We thethe demographic andand clinical characteristics of controls and patients with RA, SLERA, or Werecorded recorded demographic clinical characteristics of controls and patients with pSS, sex, anti-cyclic citrullinated (anti-CCP) positivity, and rheumatoid SLEincluding or pSS, age, including age, sex, anti-cyclicpeptides citrullinated peptides (anti-CCP) positivity,factor and positivity (Table 1). rheumatoid factor positivity (Table 1). characteristics of controls and and patients with rheumatoid arthritis, systemic lupus Table Table1.1.Demographic Demographic characteristics of controls patients with rheumatoid arthritis, systemic erythematosus, or primary Sjögren’s syndrome. lupus erythematosus, or primary Sjögren’s syndrome.

Patients with Patients with SLE Patients Patientswith withpSS pSS Controls Patients with RARA Patients with SLE Controls = 33) = 20) 20) (n (n =(n33) (n (n = 20) (n(n==20) (n ==20) 20) Age, mean ± SD (years) 62.5 ± 12.2 38.0 ± 14.0 55.2 ± 11.2 56.7 ± 13.7 Age, mean ± SD (years) 62.5 ± 12.2 38.0 ± 14.0 55.2 ± 11.2 56.7 ± 13.7 Sex (F:M) 27:6 19:1 19:1 15:5 Sex (F:M) 27:6 19:1 19:1 15:5 Anti-CCP positivity, n (%) 29 (87.9) 1 (5.0) 2 (10.0) 0 Anti-CCP positivity, n (%) 29 (87.9) 1 (5.0) 2 (10.0) 0(0) (0) positivity, n (%) 25 (75.8) 5 (25.0) 11(55.0) (55.0) 00(0) RF RF positivity, n (%) 25 (75.8) 5 (25.0) 11 (0) RA, arthritis; SLE, systemic lupus erythematosus; pSS, primary Sjögren syndrome; RA, rheumatoid rheumatoid arthritis; SLE, systemic lupus erythematosus; pSS, primary Sjögren syndrome; SD, standardSD, deviation;deviation; anti-CCP, antibodies cyclic citrullinated peptides; RF, rheumatoid factor. standard anti-CCP,toantibodies to cyclic citrullinated peptides; RF, rheumatoid factor. Variable Variable

2.2.Validation ValidationofofCarbamylaion CarbamylaionofofGRP78 GRP78 2.2. Thecarbamylated carbamylatedGRP78, GRP78,but butnot notthe thenative nativeform formGRP78, GRP78,was wasrecognizable recognizableby byanti-modified anti-modified The citrulline antibody (Figure 1). citrulline antibody (Figure 1).

Figure1.1.Verification Verificationofofthe thecarbamylated carbamylatedGRP78 GRP78(CarGRP78) (CarGRP78)by byWestern Westernblotting. blotting.Cloned ClonedGRP78 GRP78 Figure andCarGRP78 CarGRP78were were modified by 2,3-butanedione monoxime and antipyrine in aacid strong acid and modified by 2,3-butanedione monoxime and antipyrine in a strong solution. solution. The modified citrulline residueswith were detected withanti-modified rabbit polyclonal anti-modified The modified citrulline residues were detected rabbit polyclonal citrulline antibodies. citrulline antibodies. Only the CarGRP78 reacted with anti-modified Only the CarGRP78 reacted with anti-modified citrulline antibodies. citrulline antibodies.

2.3. Serum Titers of Anti-GRP78 and Anti-CarGRP78 Antibody among Controls and Patients with Rheumatoid Arthritis, Systemic Lupus Erythematosus, or Primary Sjögren’s Syndrome There were no statistically significant differences in the serum titers of anti-GRP78 antibody in patients with RA, SLE, or pSS, compared with the controls (Figure 2A). The percentage of positive

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2.3. Serum Titers of Anti-GRP78 and Anti-CarGRP78 Antibody among Controls and Patients with Rheumatoid Arthritis, Systemic Lupus Erythematosus, or Primary Sjögren’s Syndrome There were no statistically significant differences in the serum titers of anti-GRP78 antibody in Int. J. Mol. Sci. 2016, 17, 1510 3 of 10 patients with RA, SLE, or pSS, compared with the controls (Figure 2A). The percentage of positive of anti-GRP78 antibody was 10% (2/20) controls, 6% (2/33) in patients with with RA, 15% in patients of anti-GRP78 antibody was 10% in (2/20) in controls, 6% (2/33) in patients RA, (3/20) 15% (3/20) in with SLE, andwith 5% SLE, (1/20) patients pSS.with pSS. patients andin5% (1/20) inwith patients

Figure 2. Comparison of anti-GRP78 anti-carbamylatedGRP78 GRP78(CarGRP78) (CarGRP78) antibody antibody levels Figure 2. Comparison of anti-GRP78 andand anti-carbamylated levelsinin the the sera from controls, and patients with rheumatoid arthritis (RA), systemic lupus erythematosus sera from controls, and patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) or (SLE) or primary Sjögren syndrome (pSS). (A) We observed no significant differences in anti-GRP78 primary Sjögren syndrome (pSS). (A) We observed no significant differences in anti-GRP78 antibody antibody levels in the serum between patients with RA, SLE or pSS and controls; (B) Patients with levels in the serum between patients with RA, SLE or pSS and controls; (B) Patients with RA but not RA but not SLE or pSS had significantly higher sera titers of anti-CarGRP78 antibodies compared SLE or pSS had significantly higher sera titers of anti-CarGRP78 antibodies compared with controls. with controls. O.D., optical density. The cutoff values for the anti-GRP78 and anti-CarGRP78 O.D., antibody optical density. The cutoffasvalues for plus the anti-GRP78 anti-CarGRP78 antibody ELISA was ELISA was defined the mean two standardand deviations of the controls. defined as the mean plus two standard deviations of the controls. The serum levels of anti-CarGRP78 in patients with RA was significantly higher compared with the controls 0.15 ± 0.08 versus ± 0.03,with p = 0.033; Figure 2B). On the other hand, no The serum levels(OD of405anti-CarGRP78 in 0.11 patients RA was significantly higher compared significant differences were observed when comparing the serum levels of anti-CarGRP78 antibody with the controls (OD405 0.15 ± 0.08 versus 0.11 ± 0.03, p = 0.033; Figure 2B). On the other hand, in the controls with SLE or pSS patients (Figure 2B). The percentage of positive of anti-CarGRP78 no significant differences were observed when comparing the serum levels of anti-CarGRP78 antibody antibody was 0% (0/20) in controls, 27% (9/33) in patients with RA, 35% (7/20) in patients with SLE, in theand controls withinSLE or pSS (Figure 2B). The percentage of positive of anti-CarGRP78 15% (3/20) patients withpatients pSS.

antibody was 0% (0/20) in controls, 27% (9/33) in patients with RA, 35% (7/20) in patients with SLE, and 15% (3/20) in patients with pSS.

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Correlation with LevelsofofAnti-GRP78 Anti-GRP78or orAnti-CarGRP78 Anti-CarGRP78 Antibody 2.4.2.4. Correlation with Levels Antibodywith withAnti-CCP Anti-CCPAntibody Antibody found a positive correlation between serum levels of anti-GRP78 antibody the WeWe found a positive correlation between the the serum levels of anti-GRP78 antibody and and the levels levels of anti-CCP inwith patients with RA3A). (Figure 3A). However, were no significant of anti-CCP antibody antibody in patients RA (Figure However, there werethere no significant correlations correlations between the serum levels of anti-CarGRP78 antibody and the levels of between the serum levels of anti-CarGRP78 antibody and the levels of anti-CCP antibody anti-CCP in patients antibody with RA. in patients with RA.

Figure 3. Correlation between anti-CCPlevels levelsininpatients patientswith withrheumatoid rheumatoidarthritis arthritisand and their their levels levels of Figure 3. Correlation between anti-CCP of anti-GRP78 and anti-CarGRP78 antibody. (A) was There was a statistically significant positive anti-GRP78 and anti-CarGRP78 antibody. (A) There a statistically significant positive correlation correlation between the anti-CCP levels and anti-GRP78 antibody levels in patients with rheumatoid between the anti-CCP levels and anti-GRP78 antibody levels in patients with rheumatoid arthritis; arthritis; (B) There was no statistically significant correlation between the anti-CCP levels and (B) There was no statistically significant correlation between the anti-CCP levels and anti-CarGRP78 anti-CarGRP78 antibody levels in patients with rheumatoid arthritis. O.D., optical density. antibody levels in patients with rheumatoid arthritis. O.D., optical density.

2.5. Correlation with Levels of Anti-GRP78 or Anti-CarGRP78 Antibody with CRP Levels We also found that there were no significant correlations between the serum level of anti-GRP78 antibody or anti-CarGRP78 and the CRP levels in patients with RA (Figure 4).

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2.5. Correlation with Levels of Anti-GRP78 or Anti-CarGRP78 Antibody with CRP Levels Int. J. Mol. Sci. 2016, 17, 1510 We also found that there were no significant correlations between the serum level of

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anti-GRP78 antibody or anti-CarGRP78 and the CRP levels in patients with RA (Figure 4).

4. Correlation between CRP levels in patients with rheumatoid arthritis and their levels of Figure 4.Figure Correlation between CRP levels in patients with rheumatoid arthritis and their levels of anti-GRP78 and anti-CarGRP78 antibody. (A) There was no statistically significant correlation anti-GRP78 and anti-CarGRP78 antibody. (A) There was no statistically significant correlation between between the CRP levels and anti-GRP78 antibody levels in patients with rheumatoid arthritis; the CRP(B) levels and antibody levels in patients with arthritis; (B) There was There wasanti-GRP78 no statistically significant correlation between therheumatoid CRP levels and anti-CarGRP78 no statistically correlation betweenarthritis. the CRP levels and anti-CarGRP78 antibody levels in antibodysignificant levels in patients with rheumatoid O.D., optical density. patients with rheumatoid arthritis. O.D., optical density.

3. Discussion In this study, we demonstrated the presence of anti-CarGRP78 antibody in patients with RA. 3. Discussion We did not find a significant difference between patients with RA and controls in the serum titer of

In this study,antibody we demonstrated presence ofnoted anti-CarGRP78 antibody inwere patients anti-GRP78 as previously the described [2]. We that the titers of anti-GRP78 high inwith RA. few find controls, which led to an elevated cutoff value. This could by the in source of our titer of We did not a significant difference between patients with be RAexplained and controls the serum controls, which was rheumatology outpatients rather than healthy controls. In addition, the presence anti-GRP78 antibody as previously described [2]. We noted that the titers of anti-GRP78 were high of high anti-GRP78 levels could be ethnicity-related. in few controls, which led to an elevated cutoff value. This could be explained by the source of our Compared with controls, the titer of anti-CarGRP78 antibody was significantly increased only controls,inwhich was rheumatology outpatients rather than healthy controls. In addition, the presence patients with RA (p = 0.033), but not in patients with SLE or in patients with pSS. We found that of high anti-GRP78 levels could be ethnicity-related. Compared with controls, the titer of anti-CarGRP78 antibody was significantly increased only in patients with RA (p = 0.033), but not in patients with SLE or in patients with pSS. We found that the percentage of positive anti-CarGRP78 antibody was 27% in patients with RA, 35% in patients with SLE, and 15% in patients with pSS. In comparison with other studies, anti-CarP antibodies were found in 45% of Japanese patients with RA [11], 11% of patients with SLE [12], and 27% of patients with pSS [13]. Furthermore, anti-CarP antibodies could be found in serum from patients with gout, osteoarthritis, spondyloarthritis, and psoriatic arthritis, though the percentage of anti-CarP antibodies positivity was below 10% among these patients [14,15]. Thus, compared with anti-CCP, anti-CarP antibodies appeared to be less specific for a differential diagnosis of RA. Further investigations are warranted to examine the presence of anti-CarGRP78 in other systemic autoimmune diseases and arthritides.

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Currently, there are still doubts about the specificity of anti-CarP antibodies. Cross-reactivity between the anti-CarP antibodies and anti-CCP might exist [9,16]. Our study showed that there was a positive correlation between the titer of anti-CCP and anti-GRP78, but not anti-CarGRP78, suggesting that anti-CarGRP78 might be different from anti-CCP, as previously reported [17]. GRP78 is an important autoantigen for RA. GRP78 on its own or GRP78-specific regulatory T cells have been considered as new therapeutic strategies for treating RA [18,19]. In this study, we did not observe any significant associations between the CRP levels and anti-GRP78 or anti-CarGRP78. Nevertheless, it is difficult to rule out the effects of potential confounders such as disease duration and use of medication in our cross-sectional study. With the elevated anti-CarGRP78 antibody levels observed in patients with RA, we still speculated that carmabylated GRP78 could play a role in the immunopathogenesis of RA. 4. Materials and Methods 4.1. Materials Acrylamide, ammonium persulfate, ampicillin, benzamidine, calcium chloride (CaCl2 ), dithiothreitol (DTT), ethylenediaminetetraacetic acid (EDTA), glycine, imidazole, isopropyl β-D-1-thiogalactopyranoside (IPTG), Luria-Bertani (LB) broth, magnesium chloride (MgCl2 ), nickel chloride, phenylmethylsulfonyl fluoride (PMSF), polyvinylidene fluoride (PVDF) membrane, potassium chloride (KCl), sodium azide (NaN3 ), sodium bicarbonate (NaHCO3 ), sodium carbonate (Na2 CO3 ), sodium chloride (NaCl), sodium dodecyl sulfate, tetramethylethylenediamine (TEMED), tris(hydroxymethyl)aminomethane (Tris) were obtained from Sigma-Aldrich (St. Louis, MO, USA). 4.2. Patients In this study, 33 patients satisfying the 1987 American College of Rheumatology (ACR) revised criteria for the classification of RA [20], 20 patients satisfying the 1982 ACR revised criteria for the classification of SLE [21] and 20 patients satisfying the American-European Consensus Group Criteria for pSS [22] were recruited. For a strict evaluation of the clinical usefulness of the autoantibodies in the differential diagnosis of RA, instead of healthy individuals from the community, we randomly recruited 20 patients from the rheumatology outpatient clinic of our hospital with no known systemic autoimmune diseases as controls. All participants signed informed consent. The study was approved by the institutional review board of the Dalin Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Taiwan (No. B10403022, 27 October 2015). 4.3. Preparation of GRP78 and Carbamylated GRP78 Cloning of GRP78 was based on the methods described previously with modifications [23]. Full-length GRP78 cDNA was amplified by polymerase chain reaction (PCR). The resulting products were digested with NcoI and EcoRI (New England Biolabs, Ipswich, MA, USA) and then subcloned into pET32a. E. coli BL21 (DE3) was then transformed with recombinant pET-32a encoding GRP78. The transformed bacteria were grown in LB broth with ampicillin (0.1 g/L) and induced with 1 mM IPTG for 4 h at 37 ◦ C. Bacteria were harvested by centrifugation, resuspended in 100 mL of 20 mM Tris-HCl buffer (pH 7.9) containing 0.5 M NaCl, 0.2 mM PMSF, 0.02% NaN3 , 4 mM benzamidine and 0.5 mM imidazole, and lysed using a French press. GRP78 was purified from the crude lysate by sequential separation on nickel Sepharose column. The purified protein fractions were pooled, concentrated by ultrafiltration and dialyzed against polymerization buffer (5 mM Tris-HCl (pH 7.5), 2 mM CaCl2 , 0.1 M KCl, 1 mM MgCl2 , and 1 mM ATP). The purified GRP78 was stored at 4 ◦ C before use. The cloned GRP78 was carbamylated by the method described by Shi et al. [6]. The CarGRP78 was extensively dialyzed against PBS and then stored.

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4.4. Western Blotting CarGRP78 were detected by Western blotting using a modified anti-citrulline detection kit (Upstate Biotechnology, Lake Placid, NY, USA) [24]. Briefly, 20 ng CarGRP78 and GRP78 were immobilized on PVDF membrane after 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by electrotransfer. Then, immobilized CarGRP78 and GRP78 were modified by 2,3-butanedione monoxime and antipyrine in a strong acid solution, according to the manufacturer’s instructions. The modified citrulline residues were detected by the rabbit polyclonal anti-modified citrulline antibodies and goat anti-rabbit IgG-horseradish peroxidase (HRP) antibody conjugate. 4.5. Enzyme-Linked Immunosorbent Assay (ELISA) for Anti-Cyclic Citrullinated Peptides (Anti-CCP), Anti-78-kDa Glucose-Regulated Protein (Anti-GRP78) Antibody, and Anti-Carbamylated 78-kDa Glucose-Regulated Protein Anti-CarGRP78 Antibody Anti-CCP was detected by Quanta Lite CCP3 enzyme-linked immunosorbent assay (ELISA) kits (Inova Diagnostics, San Diego, CA, USA) in accordance to the manufacturer’s recommendations. ELISA plates (BD Biosciences, San Jose, CA, USA) were coated for 2 h at 37 ◦ C with 1 µg/mL GRP78 or CarGRP78 in coating buffer containing 35 mM NaHCO3 and 15 mM Na2 CO3 , pH 9.6. The plates were washed with phosphate-buffered saline (PBS) for four times and blocked with PBS containing 2% skim milk overnight. Then, Quanta Lite reagents (Inova Diagnostics, San Diego, CA, USA) were used for performing ELISA according to the supplier’s protocol. The plates were read at 405 nm on an Anthos Zenyth 3100 multimode fluorometer (Anthos Labtec Instruments GmbH, Salzburg, Austria). All samples were analyzed at the same time. The absorbance values of control wells were low in all cases. The cutoff values for the anti-GRP78 and anti-CarGRP78 antibody ELISA was defined as the mean plus two standard deviations of the controls. 4.6. Statistical Analysis All data were represented as mean ± standard deviation. Statistical significance was assessed with non-parametric Mann-Whitney U test. Linear regression analysis was conducted to evaluate the correlations between levels of anti-GRP78 or anti-CarGRP78 antibody with the levels of CRP or anti-CCP antibody. Two-tailed p < 0.05 was considered statistically significant. 5. Conclusions In conclusion, we demonstrated the presence of anti-CarGRP78 antibody in patients with RA, SLE, and pSS. The serum titer of anti-CarGRP78 antibody was significantly elevated in patients with RA compared with controls. Acknowledgments: This work was supported by a grant from Dalin Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Taiwan (DTCRD105-I-01). Author Contributions: Ming-Chi Lu and Hsien-Bin Huang conceived and designed the experiments; Hui-Chun Yu and Pei-Hsuan Lai performed the experiments; Hui-Chun Yu and Pei-Hsuan Lai analyzed the data; Ning-Sheng Lai and Hsien-Bin Huang contributed reagents/materials/analysis tools; Ming-Chi Lu and Malcolm Koo wrote the paper. All authors read and approved the final manuscript. Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations ACPA ACR anti-CarGRP78 anti-GRP78 BiP CarP CCP DTT

anti-citrullinated protein antibody American College of Rheumatology anti-carbamylated 78-kDa glucose-regulated protein anti-78-kDa glucose-regulated protein binding immunoglobulin protein carbamylated proteins cyclic citrullinated peptides dithiothreitol

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EDTA ELISA HRP IPTG LB OD PBS PCR PMSF pSS PVDF RA RF SD SDS-PAGE SLE TEMED

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ethylenediaminetetraacetic acid enzyme-linked immunosorbent assay horseradish peroxidase isopropyl β-D-1-thiogalactopyranoside Luria-Bertani optical density phosphate-buffered saline polymerase chain reaction phenylmethylsulfonyl fluoride primary Sjögren syndrome polyvinylidene fluoride rheumatoid arthritis rheumatoid factor standard deviation dodecyl sulfate-polyacrylamide gel electrophoresis systemic lupus erythematosus tetramethylethylenediamine

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